The regulation of blood clotting (hemostasis) is carried out by the interaction of various activators, inhibitors, and positive and negative feedback mechanisms. Defects in this system can lead to an imbalance in the hemostasis system and result in either a hemorrhage or thrombosis. Thrombin (factor IIa, F IIa) is a serine protease and the central enzyme of plasmatic blood clotting. The main function of thrombin consists in the induction of fibrin polymerization and is thus essential for clot formation. Thrombin is formed by activation of the enzymatically inactive precursor molecule prothrombin (factor II, F II). In order to restrict the clotting process to the site of injury, inhibitors of thrombin become activated as well. Via inhibition or by complexing the free thrombin, inhibitory factors such as, for example, antithrombin or α2-macroglobulin (α2-M) restrict and limit the coagulation process. An imbalance within the processes of thrombin formation and inhibition can lead to hypercoagulatory or hypocoagulatory states and thus to pathological clotting disorders. Thus, the measurement of thrombin formation and inhibition reveals important information about the particular clotting state of an individual patient.
Thrombin generation tests are global clotting tests, which determine the formation and inhibition of thrombin in plasma or blood. The inherent capacity, or in the case of plasma samples the plasma-intrinsic capacity, of a sample to form and inhibit enzymatically active, free thrombin is also known as the endogenous thrombin potential (ETP). Since all biological components that are contained in a test material and that influence the formation and the inhibition of thrombin affect the endogenous thrombin potential of a sample, the ETP determination is suitable both as a global test to detect a number of components of the hemostasis system and to monitor therapeutic measures. The ETP determination allows the diagnosis of hypocoagulatory and hypercoagulatory states. Further indications include hereditary and acquired coagulopathies (hemophilia, factor deficiency II, V, VII, VIII, IX, X, XI, disseminated intravascular coagulopathy) and thrombophilic risk factors (prothrombin mutation, factor V disease, protein S, protein C and antithrombin deficiency). Acquired and transient risk factors such as, for example, pregnancy, the use of oral contraceptives, and smoking are also reflected by increased ETP values. A further interesting aspect of ETP determination is the control of anticoagulation therapies. Since the capability of thrombin formation is determined directly, the clotting potential of the patient is detected independently of the anticoagulant(s) employed. Thus ETP measurement also offers a possibility of monitoring the transitional and stabilization phases of such therapies in order to avoid over-dosage and under-dosage.
Originally, for the determination of thrombin generation, a sample was treated with a prothrombin activator and aliquots were removed from the mixture at distinct time intervals. The thrombin concentration in the individual aliquots was determined by measuring the cleavage of a chromogenic thrombin substrate. Such a procedure, which is also known as the “subsampling method,” is described, for example, in Hemker et al., “A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay process.” Thromb. Haemost. 56 (1):9-17 (1986) on page 10 in the paragraph titled “Determination of the Time Course of Amidolytic Activity.”
In EP 420 332 B1, an improved method for thrombin determination is described, which allows a continuous determination of the thrombin concentration in the reaction batch, such that the removal of a number of aliquots described above can be dispensed with. When continuously determining the thrombin concentration in a reaction batch, it is essential that the thrombin substrate used is not consumed before the thrombin inhibition is complete. The use of thrombin substrates, which have kinetic properties, such that they are reacted relatively slowly, but nevertheless proportionally to the amount of thrombin present, allows for continuous determination of the thrombin concentration in a single reaction batch. For determination of thrombin generation, the conversion kinetics of a thrombin substrate are measured in a sample of coagulable blood or plasma by means of the release of a detectable signal group. Since the thrombin substrate concentration is adjusted such that the substrate cannot be completely used up in the course of the reaction, the amount of released indicator ideally behaves proportionally to the enzymatic activity of the thrombin formed in the course of the clotting reaction (see also Hemker, H. C. et al., “Continuous registration of thrombin generation in plasma, its use for the determination of the thrombin potential.” Thromb. Haemost 70(4)-617-24 (1993)).
In thrombin generation tests, small thrombin substrates of low molecular weight are customarily employed which comprise an oligopeptide to which is coupled a detectable signal group. By means of the enzymatic activity of thrombin, the bond between peptide and signal group is hydrolyzed, and the signal group is released. By means of the measurement of the signal strength, the thrombin activity can be quantified. Examples of oligopeptide substrates which, as is known, are cleaved by thrombin, are, for example, para-nitroanilide (pNA)-coupled peptides of the sequence Ala-Gly-Arg-pNA, Ala-Arg-pNA, Gly-Arg-pNA or Pro-Arg-pNA.
It is known, however, that with thrombin substrates which have a molecular size of less than 8 kD, the physiologically relevant activity of the free thrombin is measured in addition to the physiologically irrelevant activity of the α2-macro-globulin-thrombin complex (α2MT). From the measurement of the amount of released signal group over time, reaction kinetics result which, in spite of the progressive and finally complete inhibition of the free thrombin, reach no plateau phase. Instead, the reaction kinetics continue to increase. The small peptide substrates of low molecular weight are able to penetrate to the active center of the thrombin molecule through the α2-macroglobulin-thrombin complex (α2MT) and are therefore also cleaved by complexed thrombin. The amount of cleaved substrate is therefore not strictly proportional to the amount of free thrombin, but is the result of the activity of free and α2-macroglobulin-bound thrombin. Although various techniques for the calculation of the amount of free thrombin are known (e.g. EP 1 669 761 A2, WO 2004/016807 A1), these are relatively complicated in some cases. Alternative solutions that allow a direct determination of free thrombin on the basis of the experimental data are therefore desirable.
In EP 1 159 448 B1, the use of macromolecular ovalbumin-coupled thrombin substrates in a thrombin generation assay is described. Since ovalbumin-coupled thrombin substrates have a molecular size of more than 10 kDa, they are not cleaved by α2-macroglobulin-bonded thrombin, but only by free thrombin. The use of ovalbumin-coupled thrombin substrates, however, has disadvantages because technical problems occur when peptide substrates are coupled to ovalbumin when preparing the macromolecular substrate. Occasionally, the reaction solution is highly viscous, possibly on account of ovalbumin crosslinking reactions. The use of ovalbumin-coupled thrombin substrates is thus regarded as unsatisfactory because of problems in the preparation of these substrates and thus the restricted availability of the substrates. A further disadvantage in the use of protein-coupled macrosubstrates is that they cannot be added in higher concentrations, since precipitation reactions and thus turbidity can occur in the reaction batch. This is disadvantageous, in particular for test processes which are evaluated with the aid of optical methods.